Thin-layer method for identifying stone lotus seed formula granules and stone lotus pulp formula granules and preparation method of stone lotus seed formula granules and stone lotus pulp formula granules

Abstract

The invention provides a thin-layer method for identifying peucedanum praeruptorum formula granules and peucedanum praeruptorum pulp formula granules and a preparation method of the peucedanum praeruptorum pulp formula granules, a developing solvent adopted by the thin-layer method is a mixed solution of ethyl acetate, formic acid and water, the volume ratio of the ethyl acetate to the formic acid to the water is 8.5: 1: 1, and the volume ratio of the ethyl acetate to the formic acid to the water is 1.5: 1: 1. After being unfolded, the granules are dried in the air, and are inspected at 365 nm, compared with thin-layer chromatography of the stone lotus seed formula granules and the stone lotus pulp formula granules, the stone lotus seed formula granules have spots in the range of the specific shift value of 0.65-0.75, and the stone lotus pulp formula granules have no spots in the range of the specific shift value of 0.65-0.75. The thin-layer method provided by the invention can be used for rapidly and effectively identifying the stone lotus seed formula granules and the stone lotus meat formula granules, is high in stability and good in repeatability, can effectively and comprehensively reflect the conditions of the stone lotus seed formula granules and the stone lotus meat formula granules, and provides a basis for the formulation of subsequent quality standards of the stone lotus seed formula granules and the stone lotus meat formula granules.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of traditional Chinese medicine thin layer chromatography identification, and specifically provides a thin layer method for identifying lotus plumule formula granules and lotus plumula formula granules and a preparation method of lotus plumule formula granules and lotus plumula formula granules. BACKGROUND

[0002] Lotus is a common medicinal and edible plant, mainly containing alkaloids, flavonoids, glycosides, terpenes, steroids, fatty acids, proteins, minerals and vitamins and other chemical components, and has the pharmacological activities of lipid regulation and weight loss, antibacterial, anti-inflammatory, antioxidant, hemostatic and the like. Different parts of lotus including lotus plumule, lotus seed, lotus plumule, lotus seed, lotus seed skin, etc. have reported 385 compounds, of which 86 are alkaloids, 133 are flavonoids, and 166 are other compounds.

[0003] Literatures record that the original plant of lotus plumule has multiple varieties, and the dried seeds of lotus and the dried mature fruits of rostrate balanites (south snake) have been recorded as lotus plumule. Today, lotus plumule is the dried mature fruit of lotus of the family Nymphaeaceae. It has the effects of tonifying spleen and stomach, clearing heart and removing heat, and is used for treating lockjaw, diarrhea, cold and hot vomiting, food not going down, and turbid urine. Lotus plumule contains alkaloids such as lotus plumule base, isolotus plumule base, methyl lotus plumule base, and lotus plumule monobasic base, as well as starch and raffinose.

[0004] The processed product of lotus plumule is the dried mature fruit pulp of lotus of the family Nymphaeaceae (edition 1963 of Chinese Pharmacopoeia). The processing method is to grind, remove the skin and heart, and take the clean meat. It has the effects of clearing heart and promoting appetite. The processing method of lotus seed is to harvest the lotus house in autumn when the fruit matures, take out the fruit, remove the peel, and dry, or dry after removing the lotus plumule, which has the effects of tonifying spleen and stopping diarrhea, stopping leucorrhea, benefiting kidney and astringing essence, and nourishing heart and tranquilizing.

[0005] The lotus plumule and lotus plumule decoction pieces can be distinguished by means of character identification, but the lotus plumule formula granules and the lotus plumula formula granules cannot be effectively identified from the appearance. However, the lotus plumule formula granules and the lotus plumula formula granules have different medicinal effects, and after being delivered from the factory or purchased from the pharmacy, they need to be distinguished and identified to control the quality of the medicine and realize accurate medication. However, there is no report on the identification method of the two in the prior art, so it is of great application value to establish a method for distinguishing the lotus plumule formula granules and the lotus plumula formula granules. SUMMARY

[0006] The present application aims to at least solve one of the technical problems in the prior art to some extent. To this end, one object of the present application is to provide a thin layer method for identifying lotus plumule formula granules and lotus plumula formula granules and a preparation method of lotus plumule formula granules and lotus plumula formula granules.

[0007] The present application provides a thin layer method for identifying Rhizoma Bletillae granules and Rhizoma Bletillae meat granules, wherein the developing agent is a mixed solution of ethyl acetate, formic acid and water, the volume ratio of the ethyl acetate, the formic acid and the water is 8.5:1:1, and the thin layer method is observed under 365 nm after drying.

[0008] The thin layer method for identifying Rhizoma Bletillae granules and Rhizoma Bletillae meat granules provided by the present application can be used for quickly and effectively identifying Rhizoma Bletillae granules and Rhizoma Bletillae meat granules by selecting the developing agent with a volume ratio of ethyl acetate, formic acid and water being 8.5:1:1, and taking whether there are significant spots in the range of 0.65-0.75 of the Rf value of the thin layer chromatogram as the identification point. Specifically, in the above Rf value range, Rhizoma Bletillae granules have spots, and Rhizoma Bletillae meat granules have no spots. At the same time, taking Rhizoma Bletillae medicinal materials as the control medicinal materials, the thin layer chromatogram of Rhizoma Bletillae medicinal materials and Rhizoma Bletillae granules has small differences, good separation degree and high reliability under the developing of the developing agent, and Rhizoma Bletillae medicinal materials have significant spots in the range of 0.65-0.75 of the Rf value. In addition, compared with the current method for identifying Rhizoma Bletillae medicinal materials by thin layer chromatography, the present application can be observed under 365 nm at room temperature after developing, which greatly simplifies the operation steps and improves the identification efficiency. Therefore, the thin layer method of the present application can quickly and effectively identify Rhizoma Bletillae granules and Rhizoma Bletillae meat granules, and the method has high stability and good repeatability, and can effectively and comprehensively reflect the status of Rhizoma Bletillae granules and Rhizoma Bletillae meat granules, thereby providing a basis for the subsequent development of quality standards.

[0009] In some embodiments of the present application, the sample preparation process of the Rhizoma Bletillae control medicinal materials includes: decocting the Rhizoma Bletillae control medicinal materials with water, filtering, evaporating the filtrate to dryness, extracting with 20-40 vt% methanol aqueous solution, and then filtering, and the filtrate is the sample solution of the control medicinal materials.

[0010] Those skilled in the art can understand that the above Rhizoma Bletillae granules and Rhizoma Bletillae meat granules are prepared by using Rhizoma Bletillae extract and Rhizoma Bletillae meat extract respectively, and therefore the thin layer method provided by the present application can also be used for identifying Rhizoma Bletillae extract and Rhizoma Bletillae meat extract.

[0011] In some embodiments of the present application, the sample preparation process of the Rhizoma Bletillae control medicinal materials includes: decocting the Rhizoma Bletillae control medicinal materials with water, filtering, evaporating the filtrate to dryness, extracting with 20-40 vt% methanol aqueous solution, and then filtering, and the filtrate is the sample solution of the control medicinal materials.

[0012] In some embodiments of the present application, the developing plate used in the thin layer method includes silica gel GF254. 254 Thin layer plate, silica gel G thin layer plate.

[0013] In some embodiments of the present application, the method for preparing the sample for spotting of the formula granules of Pteridophyllum racemosum (Wall.) Diels comprises: extracting the formula granules of Pteridophyllum racemosum (Wall.) Diels with 20-40 vt% methanol solution, and then filtering.

[0014] In some embodiments of the present application, the mass of the formula granules of Pteridophyllum racemosum (Wall.) Diels to the volume of the methanol solution is 1 g: (20-30) ml.

[0015] In some embodiments of the present application, the extraction is ultrasonic extraction for 20-40 min.

[0016] In some embodiments of the present application, the method for preparing the sample for spotting of the formula granules of Pteridophyllum racemosum (Wall.) Diels comprises: extracting the formula granules of Pteridophyllum racemosum (Wall.) Diels with 20-40 vt% methanol solution, and then filtering.

[0017] In some embodiments of the present application, the mass of the formula granules of Pteridophyllum racemosum (Wall.) Diels to the volume of the methanol solution is 1 g: (20-30) ml.

[0018] In some embodiments of the present application, the extraction is ultrasonic extraction for 20-40 min.

[0019] In some embodiments of the present application, the spotting amount of the sample for spotting of the formula granules of Pteridophyllum racemosum (Wall.) Diels and the formula granules of Pteridophyllum racemosum (Wall.) Diels is independently 15-25 μl, preferably 20 μl.

[0020] In some embodiments of the present application, the ambient temperature of the development of the developing agent is 4-25℃.

[0021] In some embodiments of the present application, the ambient humidity of the development of the developing plate in the thin layer method is 32-75%.

[0022] In the second aspect of the present application, the present application provides a method for preparing the formula granules of Pteridophyllum racemosum (Wall.) Diels in the above-mentioned thin layer method for identifying the formula granules of Pteridophyllum racemosum (Wall.) Diels and the formula granules of Pteridophyllum racemosum (Wall.) Diels, which comprises: boiling the Pteridophyllum racemosum (Wall.) Diels decoction pieces with water, and reducing pressure to concentrate the obtained water extract to obtain a concentrated solution; spray drying the concentrated solution to obtain Pteridophyllum racemosum (Wall.) Diels spray-dried powder, mixing the Pteridophyllum racemosum (Wall.) Diels spray-dried powder and malt dextrin uniformly to perform dry granulation.

[0023] In some embodiments of the present application, the boiling comprises first boiling and second boiling, the water addition amount of the first boiling is 8-12 times of the Pteridophyllum racemosum (Wall.) Diels decoction pieces, the time of the first boiling is 40-60 min, the water addition amount of the second boiling is 6-10 times of the Pteridophyllum racemosum (Wall.) Diels decoction pieces, the time of the second boiling is 20-40 min, and the boiling temperature is 90-100℃.

[0024] In some embodiments of the present application, the temperature of the reduced pressure concentration is 60-80℃, the vacuum degree is -0.050~-0.080Mpa, and the reduced pressure concentration is to a density of 1.08~1.10.

[0025] In some embodiments of the present application, the inlet air temperature of the spray drying is 185-195℃, and the outlet air temperature is 85-95℃.

[0026] In some embodiments of the present application, the addition amount of the malt dextrin is 5-10%.

[0027] In a third aspect of the present application, the present application provides a preparation method of the stone parsley flesh formula granules in the above-mentioned thin layer method for identifying the stone parsley fruit formula granules and the stone parsley flesh formula granules, which comprises: The stone parsley flesh decoction pieces are boiled with water, and the obtained water extract is reduced pressure concentrated to obtain a concentrated solution; The concentrated solution is spray dried to obtain stone parsley flesh spray dried powder, The stone parsley flesh spray dried powder and malt dextrin are uniformly mixed for dry granulation.

[0028] In some embodiments of the present application, the boiling comprises first boiling and second boiling, the water addition amount of the first boiling is 8-12 times of the stone parsley flesh decoction pieces, the time of the first boiling is 40-60min, the water addition amount of the second boiling is 6-10 times of the stone parsley flesh decoction pieces, the time of the second boiling is 20-40min, and the boiling temperature is 90-100℃.

[0029] In some embodiments of the present application, the temperature of the reduced pressure concentration is 60-80℃, the vacuum degree is -0.050~-0.080Mpa, and the reduced pressure concentration is to a density of 1.08~1.10.

[0030] In some embodiments of the present application, the inlet air temperature of the spray drying is 185-195℃, and the outlet air temperature is 85-95℃.

[0031] In some embodiments of the present application, the addition amount of the malt dextrin is 5-10%.

[0032] Compared with the prior art, the present application can at least realize one of the following beneficial effects: (1) The present application selects the developing agent with a volume ratio of ethyl acetate, formic acid and water of 8.5:1:1, the thin layer chromatogram of the stone parsley fruit medicinal material and the stone parsley fruit formula granules has small difference, good separation degree and high reliability; the thin layer chromatogram is in the range of specific migration value of 0.65-0.75, the stone parsley fruit formula granules have spots (the stone parsley fruit medicinal material has spots), and the stone parsley flesh formula granules have no spots, and the presence or absence of the above characteristic spots can quickly, accurately and effectively identify the stone parsley fruit formula granules and the stone parsley flesh formula granules.

[0033] (2) The thin layer method provided by the application has high stability and good repeatability, can effectively and comprehensively reflect the conditions of the stone lotus seed formula granules and the stone lotus meat formula granules, and provides a basis for subsequent quality standard formulation. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 The thin layer chromatogram of Example 1; Figure 2 The thin layer chromatogram of Example 2; Figure 3 The chromatogram of Qingdao marine thin layer plate after development of Example 3; Figure 4 The chromatogram of Germany Merck thin layer plate after development of Example 3; Figure 5 The chromatogram of Tianjin Silida thin layer plate after development of Example 3; Figure 6 The thin layer chromatogram of Example 4; Figure 7 The thin layer chromatogram of Example 5; Figure 8 The thin layer chromatogram of Example 6; Figure 9 The thin layer chromatogram of Comparative Example 1; Figure 10 The thin layer chromatogram of Comparative Example 2; Figure 11 The thin layer chromatogram of Comparative Example 3; Figure 12 The thin layer chromatogram of Comparative Example 4. DETAILED DESCRIPTION

[0035] All other examples obtained by a person of ordinary skill in the art based on the examples in the application without creative labor fall within the scope of protection of the application. The application is described below with reference to specific examples, and it should be noted that these examples are merely descriptive and do not limit the application in any way.

[0036] The material models used in the examples and comparative examples of the application are as follows: Ultrasonic machine, heating plate, mortar, thin layer imaging system: CAMAG TLC Visualizer, silica gel GF254 thin layer plate (Tianjin Silida Technology Co., Ltd., batch number: 191026, Germany Merck Company, batch number: HX87183353).

[0037] Methanol, ethyl acetate and formic acid are all analytical pure, and water is ultrapure water (laboratory self-made).

[0038] Reference material of stone lily seed (Lei Meitian Medicine and Biological Technology Co., Ltd., batch number: DSTYS003101). Stone lily seed formula granules (Sichuan New Green Pharmaceutical Technology Development Co., Ltd., batch numbers: 2302057, 2302058, 2302059).

[0039] Stone lily flesh formula granules (Sichuan New Green Pharmaceutical Technology Development Co., Ltd., batch numbers: 2507001, 2507002, 2507003).

[0040] The preparation methods of stone lily seed formula granules and stone lily flesh formula granules are as follows: 1. Processing: Take stone lily seed medicinal materials, remove impurities, crush when needed, and obtain stone lily seed decoction pieces.

[0041] Take stone lily seed medicinal materials, break open, remove the skin and core, and take the clean meat to obtain stone lily flesh decoction pieces.

[0042] 2. Extraction and concentration Take 1000g of stone lily seed decoction pieces and stone lily flesh decoction pieces, add 10 times water for the first decoction, soak for 20 minutes, and maintain decoction for 40 minutes; add 8 times water for the second decoction, and decoct for 30 minutes. The temperature is controlled at 90-100℃ during the decoction process to obtain water extract.

[0043] Take the water extract, and under the conditions of temperature 60-80℃ and vacuum degree -0.050~-0.080 Mpa, reduce pressure and concentrate to a density of 1.08~1.10 to obtain stone lily seed concentrated liquid and stone lily flesh concentrated liquid.

[0044] 3. Drying Take the concentrated liquid, and under the conditions of inlet air temperature 190±5℃ and outlet air temperature 90±5℃, perform spray drying to obtain stone lily seed spray dried powder and stone lily flesh spray dried powder.

[0045] 4. Granule preparation Take the spray dried powder, add an appropriate amount of malt dextrin, mix well, and perform dry granulation to obtain stone lily seed formula granules and stone lily flesh formula granules.

[0046] Example 1 (1) Preparation of stone lily seed reference material sample, stone lily seed formula granule sample, and stone lily flesh formula granule sample.

[0047] Preparation of stone lily seed reference material sample: 1g of stone lily seed reference material was added with 100ml of water, decocted for 60 minutes, filtered, and the filtrate was evaporated to dryness. The residue was added with 5ml of 30% methanol, ultrasonically treated for 30 minutes, filtered, and the filtrate was used as the stone lily seed reference material sample.

[0048] Preparation of spotting sample of lotus seed formula granules: Grind about 0.2g of lotus seed formula granules into a fine powder, add 5ml of 30% methanol, sonicate for 30 minutes, filter, and use the filtrate as the spotting sample of lotus seed formula granules.

[0049] Preparation of spotting sample of lotus seed formula granules: Grind about 0.2g of lotus seed formula granules into a fine powder, add 5ml of 30% methanol, sonicate for 30 minutes, filter, and use the filtrate as the spotting sample of lotus seed formula granules.

[0050] (2) According to the thin-layer chromatography method (General Chapter 0502 of the Chinese Pharmacopoeia 2020), 5 μl, 10 μl, 15 μl, 20 μl, and 25 μl of the sample solution of the reference herb *Nelumbo nucifera* seed, and 10 μl, 15 μl, 20 μl, and 25 μl of the sample solution of the *Nelumbo nucifera* seed formula granules and the sample solution of the *Nelumbo nucifera* pulp formula granules were spotted onto silica gel GF. 254 On the thin-layer plate, ethyl acetate-formic acid-water (volume ratio 8.5:1:1) was used as the developing solvent. The plate was then removed, dried, and examined at 365 nm.

[0051] For details, please see [link / details]. Figure 1 , Figure 1 In the table, samples 1-5 represent the reference material samples of *Nelumbo nucifera* seeds (5µl, 10µl, 15µl, 20µl, 25µl, respectively); sample 6 represents the reference material sample of *Nelumbo nucifera* seeds (10µl); samples 7-10 represent the granulated formulation samples of *Nelumbo nucifera* seeds (10µl, 15µl, 20µl, 25µl, respectively); and samples 11-14 represent the granulated formulation samples of *Nelumbo nucifera* kernels (10µl, 15µl, 20µl, 25µl, respectively). Figure 1 It can be seen that when the sample volume of the test samples (lotus seed formula granules and lotus pulp formula granules) is 15~25μl, the thin-layer chromatographic spots are clearly visible, and the sample volume of the reference herb solution and the test sample solution is determined to be 20μl. Compared with the chromatogram of the reference herb, the ratio shift (R0) is significantly higher. f At a value of 0.75, the lotus seed formula granules have the same spot, while the lotus pulp formula granules have no significant spots. This spot is used as the identification point for distinguishing between lotus seed formula granules and lotus pulp formula granules.

[0052] Example 2 (Specificity Investigation) The only difference between Example 2 and Example 1 is that the sample volume of the negative control solution (30% methanol), *Nelumbo nucifera* seed reference material, *Nelumbo nucifera* seed formula granules, and *Nelumbo nucifera* pulp formula granules was 20 μl. The negative control solution, *Nelumbo nucifera* seed reference material sample solution, *Nelumbo nucifera* seed formula granule sample solution, and *Nelumbo nucifera* pulp formula granule sample solution were respectively spotted onto the same silica gel GF. 254 On the thin-layer plate, ethyl acetate-formic acid-water (volume ratio 8.5:1:1) was used as the developing solvent. The plate was then removed, dried, and examined at 365 nm.

[0053] For details, please see [link / details]. Figure 2 , Figure 2 In the table, 1 and 6 are negative controls; 2 and 7 are *Nelumbo nucifera* seed reference materials; 3-5 are *Nelumbo nucifera* seed formula granules; and 8-10 are *Nelumbo nucifera* kernel formula granules. Figure 2 It can be seen that the negative sample did not interfere with the test samples of lotus seed formula granules and lotus pulp formula granules, indicating that the method has good specificity. Compared with the chromatogram of lotus seed reference material, the ratio shift (R0) was significantly higher. f At a value of 0.73, the same spots were observed in the lotus seed formula granules, while the lotus pulp formula granules showed no significant spots.

[0054] Example 3 (Durability Study) The only difference between Example 3 and Example 1 is that the sample volume for the lotus seed control material, lotus seed formula granules, and lotus pulp formula granules is 20 μl, and pre-formulated silica gel GF from three brands—Merck (Germany), Tianjin Slida, and Qingdao Haiyang—is used. 254 Thin-layer plate.

[0055] The chromatogram of the Qingdao marine thin-layer plate after development is shown below. Figure 3 , Figure 3 In the table, 1 and 4 are reference materials of *Nelumbo nucifera* seeds; 2-3 are granulated formulations of *Nelumbo nucifera* seeds; and 5-6 are granulated formulations of *Nelumbo nucifera* kernels. The chromatograms after development on a Merck thin-layer plate are shown below. Figure 4 , Figure 4 In the table, 1 and 4 are reference materials of *Nelumbo nucifera* seeds; 2-3 are formula granules of *Nelumbo nucifera* seeds; 5-6 are formula granules of *Nelumbo nucifera* kernels; the chromatograms after thin-layer plate development by Tianjin Silida Technology Co., Ltd. are shown below. Figure 5 , Figure 5 In the table, 1 and 4 are reference materials for *Nelumbo nucifera* seeds; 2-3 are *Nelumbo nucifera* seed formula granules; and 5-6 are *Nelumbo nucifera* kernel formula granules. Figures 3-5 It can be seen that all three brands of thin-layer chromatography plates can meet the expected identification requirements, indicating that the method has good durability. Compared with the reference material chromatogram of lotus seed, the ratio shift (R0) of the Merck thin-layer chromatography plate from Germany is higher. f At a value of 0.72, the same spots were observed in the lotus seed formulation granules, while no significant spots were observed in the lotus pulp formulation granules; the specific displacement value (R0.72) of the Tianjin Silida thin-layer plate was... f At a value of 0.66, the same spots were observed in the lotus seed formulation granules, while no significant spots were observed in the lotus pulp formulation granules; the specific displacement value (R0.66) of the Qingdao marine thin-layer plate was also observed. f At a value of 0.70, the lotus seed formula granules had the same spots, while the lotus pulp formula granules had no significant spots.

[0056] Example 4 (Investigation at different temperatures) The only difference between Example 4 and Example 1 is that the sample volume of the lotus seed control material, lotus seed formula granules, and lotus seed pulp formula granules is 20 μl. The thin-layer plates after sampling are taken and developed at low temperature of 4℃ and normal temperature of 25℃, respectively.

[0057] For details, please see [link / details]. Figure 6 , Figure 6 In the table, 1 and 4 are reference materials for *Nelumbo nucifera* seeds; 2-3 are *Nelumbo nucifera* seed formula granules; and 5-6 are *Nelumbo nucifera* kernel formula granules. Figure 6 It can be seen that this method has good durability under different temperatures. Compared with the reference chromatogram of lotus seed, at a temperature of 4℃, the ratio shift (R0) is significantly higher. f At a value of 0.73, the same spots were observed in the lotus seed formulation granules, while no significant spots were observed in the lotus pulp formulation granules; at a temperature of 25℃, the specific displacement value (R0) was... f At a value of 0.66, the same spots were observed in the lotus seed formula granules, while the lotus pulp formula granules showed no significant spots.

[0058] Example 5 (Investigation at different humidity levels) The only difference between Example 5 and Example 1 is that the sample volume of the lotus seed control material, lotus seed formula granules, and lotus pulp formula granules is 20 μl. The thin-layer plates after sampling are developed in 32% and 75% humidity environments, respectively.

[0059] For details, please see [link / details]. Figure 7 , Figure 7 In the table, 1 and 4 are reference materials for *Nelumbo nucifera* seeds; 2-3 are *Nelumbo nucifera* seed formula granules; and 5-6 are *Nelumbo nucifera* kernel formula granules. Figure 7 It can be seen that this method has good durability under different humidity levels. Compared with the reference medicinal material atlas of lotus seeds, when the humidity is 32%, the ratio shift (R0) is significantly higher. f At a value of 0.65, the lotus seed formulation granules showed the same spots, while the lotus pulp formulation granules showed no significant spots; when the humidity was 75%, the specific displacement value (R) was... f At a value of 0.66, the same spots were observed in the lotus seed formula granules, while the lotus pulp formula granules showed no significant spots.

[0060] Example 6 The thin-layer chromatography method of Example 1 was used, with a sample size of 20 μl. Three batches of lotus seed formulation granules and three batches of lotus pulp formulation granules were used to verify the thin-layer chromatography method.

[0061] For details, please see [link / details]. Figure 8 , Figure 8 In the diagram, 1 and 5 represent the reference material of *Nelumbo nucifera* seeds; 2-4 represent the formulated granules of *Nelumbo nucifera* seeds; and 6-8 represent the formulated granules of *Nelumbo nucifera* kernels. The results showed that compared with the chromatogram of the reference material of *Nelumbo nucifera* seeds, the ratio shift (R0) was significantly higher. fAt a value of 0.72, the same spots were observed in the lotus seed formula granules, while the lotus pulp formula granules showed no significant spots.

[0062] The ratio shift values ​​of the identification spots in Examples 1, 2-6 were summarized, and the ratio shift values ​​of various data in the thin-layer chromatography method investigation were summarized. The results are shown in Table 1. Based on the summarized results, the ratio shift values ​​(R0) of the identification spots of the lotus seed and lotus pulp formulation granules were determined. f The percentage should be within 10%, with an average of 0.70.

[0063] Table 1

[0064] Comparative Example 1 The only difference between Comparative Example 1 and Example 1 is as follows: Provincial standard method: Take 1g of this product, grind it finely, add 10ml of 70% ethanol, sonicate for 30 minutes, filter, and use the filtrate as the test solution. Separately, take 1g of *Nelumbo nucifera* seed reference material, add 100ml of water, boil, maintain a gentle boil for 1 hour, cool, filter, evaporate the filtrate to dryness, add 10ml of 70% ethanol to the residue, and prepare the reference material solution using the same method. Perform thin-layer chromatography (Chinese Pharmacopoeia 2020 Edition, General Chapter 0502), taking 5μl of the test solution and 10μl of the reference material solution, and spotting them separately on the same silica gel GF254 thin-layer plate. Use n-butanol-isopropanol-water-glacial acetic acid (7:5:1:1) as the developing solvent, develop, remove, air dry, and examine under ultraviolet light (365nm). In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatogram of the reference material.

[0065] For details, please see [link / details]. Figure 9 , Figure 9 In the table, 1 represents the reference medicinal material of lotus seed, 2 represents the formula granules of lotus seed, and 3 represents the formula granules of lotus pulp. Figure 9 It can be seen that, compared with the reference medicinal material atlas of lotus seeds, there are no obvious differences in the spots between the lotus seed formula granules and the lotus seed pulp formula granules, and the two cannot be distinguished.

[0066] Comparative Example 2 The only difference between Comparative Example 2 and Example 1 is that the sample amount of the lotus seed control material, lotus seed formula granules, and lotus pulp formula granules was 20 μl, and the developing solvent was n-hexane-acetone (7:2).

[0067] For details, please see [link / details]. Figure 10 , Figure 10 In the table, 1 represents the reference medicinal material of lotus seed, 2 represents the formula granules of lotus seed, and 3 represents the formula granules of lotus seed kernel. Figure 10 It can be seen that no obvious dots are found in the atlas, so the two cannot be distinguished.

[0068] Comparative Example 3 The only difference between Comparative Example 3 and Example 1 is that the sample amount of the lotus seed control material, lotus seed formula granules, and lotus pulp formula granules was 20 μl, and the developing solvent was chloroform-ethyl acetate-diethylamine (5:4:1).

[0069] For details, please see [link / details]. Figure 11 , Figure 11 In the table, 1 represents the reference medicinal material of lotus seed, 2 represents the formula granules of lotus seed, and 3 represents the formula granules of lotus seed kernel. Figure 11 It can be seen that, compared with the reference medicinal material atlas of lotus seeds, there are no obvious differences in the spots between the lotus seed formula granules and the lotus seed pulp formula granules, and the two cannot be distinguished.

[0070] Comparative Example 4 The only difference between Comparative Example 4 and Example 1 is that the sample amount of the reference herb *Nelumbo nucifera*, the formula granules of *Nelumbo nucifera*, and the formula granules of *Nelumbo nucifera* kernel was 20 μl, and the developing solvent was ethyl acetate-acetic acid-water (8.5:1:1).

[0071] For details, please see [link / details]. Figure 12 , Figure 12 In the table, 1 represents the reference medicinal material of lotus seed, 2 represents the formula granules of lotus seed, and 3 represents the formula granules of lotus seed kernel. Figure 12 It can be seen that, compared with the reference medicinal material atlas of lotus seeds, there are no obvious differences in the spots between the lotus seed formula granules and the lotus seed pulp formula granules, and the two cannot be distinguished.

[0072] Any numerical value mentioned in this invention, if there is only a two-unit interval between any minimum and any maximum value, includes all values ​​that increase by one unit each time from the minimum to the maximum value. For example, if the amount of a component, or the value of a process variable such as temperature, pressure, or time, is stated as 50-90, in this specification it means specifically listing values ​​such as 51-89, 52-88… and 69-71 and 70-71, etc. For non-integer values, it may be appropriately considered that a unit is 0.1, 0.01, 0.001, or 0.0001. These are merely some specifically specified examples. In this application, in a similar manner, all possible combinations of numerical values ​​between the listed minimum and maximum values ​​are considered to have been disclosed.

[0073] It should be noted that the embodiments described above are only for explaining the present invention and do not constitute any limitation on the present invention. The present invention has been described with reference to typical embodiments, but it should be understood that the words used therein are descriptive and explanatory terms, not limiting terms. Modifications can be made to the present invention within the scope of the claims, and revisions can be made to the present invention without departing from the scope and spirit of the present invention. Although the present invention described herein relates to specific methods, materials, and embodiments, it does not mean that the present invention is limited to the specific examples disclosed herein; on the contrary, the present invention can be extended to all other methods and applications with the same function.

Claims

1. A thin-layer chromatography method for distinguishing between lotus seed formulation granules and lotus pulp formulation granules, characterized in that, The thin-layer chromatography method uses a mixed solution of ethyl acetate, formic acid, and water as the developing solvent. The volume ratio of ethyl acetate, formic acid, and water is 8.5:1:

1. After development and drying, the solution is examined at 365 nm. Compared with the thin-layer chromatography of lotus seed granules, lotus seed granules showed spots in the range of 0.65-0.75, while lotus seed granules showed no spots in the range of 0.65-0.

75.

2. The method according to claim 1, characterized in that, The thin-layer method uses a developing plate including silicone GF. 254 Thin-layer plates, silicone G thin-layer plates; And / or, the spotting volume of the lotus seed formula granules and the spotting volume of the lotus pulp formula granules are each independently 15~25μl, preferably 20μl; And / or, the ambient temperature for the development of the developing agent is 4℃-25℃; And / or, the ambient humidity during the unfolding of the unfolding plate in the thin-layer method is 32%-75%.

3. The method according to claim 1 or 2, characterized in that, The preparation method of the spotting sample of the lotus seed formula granules includes: extracting the lotus seed formula granules with a 20-40 wt% methanol aqueous solution, and then filtering.

4. The method according to claim 3, characterized in that, The mass ratio of the lotus seed formula granules to the volume ratio of the methanol aqueous solution is 1g:(20-30)ml; And / or, the extraction is ultrasonic extraction for 20-40 minutes.

5. The method according to claim 1 or 2, characterized in that, The preparation method of the spotting sample of the lotus seed formula granules includes: extracting the lotus seed formula granules with a 20-40 wt% methanol aqueous solution, and then filtering.

6. The method according to claim 5, characterized in that, The mass ratio of the lotus seed extract granules to the volume ratio of the methanol aqueous solution is 1g:(20-30)ml; And / or, the extraction is ultrasonic extraction for 20-40 minutes.

7. A method for preparing lotus seed formulation granules in a thin-layer chromatography method for distinguishing lotus seed formulation granules from lotus pulp formulation granules according to any one of claims 1-6, characterized in that, include: Take lotus seed slices, add water and decoct, then concentrate the resulting aqueous extract under reduced pressure to obtain a concentrated solution. The concentrated solution was spray-dried to obtain spray-dried lotus seed powder. The spray-dried lotus seed powder and maltodextrin are mixed and then dry granulated.

8. The method according to claim 7, characterized in that, The decoction process includes a first decoction and a second decoction. The amount of water added in the first decoction is 8-12 times that of the lotus seed slices, and the first decoction time is 40-60 minutes. The amount of water added in the second decoction is 6-10 times that of the lotus seed slices, and the second decoction time is 20-40 minutes. The decoction temperature is 90-100℃. And / or, the vacuum concentration temperature is 60-80℃, the vacuum degree is -0.050~-0.080Mpa, and the vacuum concentration is carried out to a density of 1.08~1.10; And / or, the inlet air temperature of the spray dryer is 185-195℃, and the outlet air temperature is 85-95℃; And / or, the amount of maltodextrin added is 5-10%.

9. A method for preparing lotus seed extract granules in a thin-layer chromatography method for distinguishing lotus seed extract granules from lotus pulp extract granules according to any one of claims 1-6, characterized in that, include: Take the lotus seed slices, add water and decoct. Concentrate the resulting aqueous extract under reduced pressure to obtain a concentrated solution. The concentrated solution was spray-dried to obtain spray-dried lotus seed powder. The spray-dried lotus seed powder and maltodextrin were mixed and then dry granulated.

10. The method according to claim 9, characterized in that, The decoction process includes a first decoction and a second decoction. The amount of water added in the first decoction is 8-12 times that of the lotus seed slices, and the first decoction time is 40-60 minutes. The amount of water added in the second decoction is 6-10 times that of the lotus seed slices, and the second decoction time is 20-40 minutes. The decoction temperature is 90-100℃. And / or, the vacuum concentration temperature is 60-80℃, the vacuum degree is -0.050~-0.080Mpa, and the vacuum concentration is carried out to a density of 1.08~1.10; And / or, the inlet air temperature of the spray dryer is 185-195℃, and the outlet air temperature is 85-95℃; And / or, the amount of maltodextrin added is 5-10%.

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